1. Field of the Invention
The present invention relates to microbiological industry, to the method of L-arginine production and concerns the using of new feedback-resistant mutant enzymes in arginine biosynthesis pathway of E. coli arginine-producer strains.
2. Description of the Related Art
The biosynthesis of arginine from glutamate in E. coli cells is carried out by a series of reactions initiated by the acetylation of glutamate by N-acetylglutamate synthase (NAGS) encoded by argA. This process is regulated via transcription repression of the arg regulon and by feedback inhibition of NAGS by arginine [Cunin R., et al., Microbiol. Rev., vol. 50, p. 314–352, 1986]. L-Arginine represses argA expression with a ratio greater than 250 and inhibits NAGS activity (Ki=0.02 mm) [Leisinger T., Haas D., J. Biol. Chem., vol. 250, p. 1690–1693, 1975]. For enhanced biosynthesis of arginine in E. coli, the feedback-resistant (may be referred to as “fbr”) NAGS enzymes are required.
The feedback-resistant mutants of enzymes can be obtained by spontaneous, chemical or site-directed mutagenesis.
Some argA fbr mutants were isolated and studied. The Serratia marcescens cells carrying the chromosomal fbr argA mutations were unstable and gave rise to argA mutants with reduced activity or with altered affinity for glutamate [Takagi T., et al., J.Biochem. vol. 99, p. 357–364 1986].
The fbr argA genes from the five E. coli strains with fbr NAGS were cloned and different single-base substitutions in argA genes were found in each of the fbr NAGS strains and it was revealed that the substitutions cause replacing His-15 with Tyr, Tyr-19 with Cys, Ser-54 with Asn, Arg-58 with His, Gly-287 with Ser and Gln-432 with Arg (Rajagopal B. S. et al., Appl. Environ. Microbiol., 1998, vol. 64, No. 5, p. 1805–1811).
As a rule, the fbr phenotype of enzyme arises as a result of the replacing the amino acid residue with another in a single or in a few sites of protein sequence and these replacements lead to reducing the activity-of enzyme. For example, the replacing of natural Met-256 with 19 other amino acid residues in E. coli serine acetyltransferase (SAT) (cysE gene) leads in most cases to fbr phenotype but the mutant SAT proteins do not restore the level of activity of natural SAT (Nakamori S. et al., AEM, 64(5):1607–11, 1998).
So, the disadvantage of the mutant enzymes, obtained by these methods, is a reduce in the activity of mutant enzymes as compared to wild type enzymes.